Beta-glucosidase



A β-glucosidase is an enzyme which catalyses the hydrolysis of terminal non-reducing residues in β-glucosides (EC number : 3.2.1.21). In the case of 2VRJ, it comes from Thermotoga maritima which is a rod-shaped bacterium belonging to the order of Thermotogates. This bacterium was originally isolated from geothermal heated marine sediments. 2VRJ is here is in complex with an inhibitor called N-octyl-5-deoxy66-oxa-N-carbamoylcalystegine.

General action as biocatalyst
It acts on the β(1-4) bond linking two glucose residues or glucose-substituted molecules. The action of the enzyme on such glucosides results in the release of units of glucose. For instance, hydrolysis of cellobiose catalysed by a β-glucosidase releases two glucoses.



β-glucosidases can also be called β-D-glucoside glucohydrolases or cellobiases.

Structure and function
 In terms of structure 2VRJ is a homodimer. It means that it is composed of two chains A and B which are chiral. Each chain is composed of 438 residues and constitutes a subunit of the protein. Each subunit contains a catalytic site. The enzymatic hydrolysis of a glycosidic bond requires two critical residues : a proton donor and a proton acceptor which can also be called a nucleophile/base. Aspartate and glutamate have been found to perform catalysis. Accorded to this, studies showed that one of the conserved regions of β-glucosidases is centred on conserved glutamate residues. As every β-glucosidase, 2VRJ presents two conserved residues of glutamate (166 and 351 ). Moreover 2VRJ has a third important residue : asparagin 293. The protein is presented in complex with an inhibitor called calystegine. We can see that the two glutamate  residues and the asparagin are really closed to each other and to the ligand. Such a proximity highly suggests that there are important interactions between them. So we can say that the catalytic site of 2VRJ is composed of two glutamate and one asparagin.

There are three different topologies for the active site of β-glucosidases : the pocket or crater, the cleft or groove and the tunnel. The topology of 2VRJ active site is a pocket in which the ligand can bind. 

Hydrolysis of terminal non-reducing residues in β-glucosides
There are two ways to hydrolyse the terminal non-reducing residues in β-glucosides which implicate the two glutamate residues and a molecule of water. Water which is an amphoter, is here used as a base for the nucleophilic attack on the positively charged anomeric carbon.

The general equation of the chemical reaction is :

Inverting glycoside hydrolases
Inverting glycoside hydrolases lead to an inversion of the anomeric configuration to create an alpha configuration. The steps of the reaction are like the mechanism of nucleophilic substitution S2N. It is an one step process: the nucleophile (water) the anomeric carbon with simultaneous expulsion of the leaving group (OR). Bond making takes place at the same time as bond breaking. Such a mechanism is called concerted reaction. The distance between the two carboxylates is about 10.5 angströms.

Retaining glycoside hydrolases
Retaining glycoside hydrolyses occur in two steps: the first step, called glycosylation leads to the release of the leaving group and the creation of a carbocation. Subsequently, water attacks this last one. The second step, called deglycosylation consists of OR- nucleophilic attack on the intermediate and allows the deglycosylation of the enzyme. In this case, there are two transition states involved. The distance between the two carboxylates for this mechanism is about 5.5 angströms. For 2VRJ the distance between its two glutamates is about 5 angströms. So we can say that 2VRJ seems to be a retaining enzyme.

NB: The values of the pH and the nature of the solvent play a main role in the rate of the reaction.

Glutamates are directly involved in the catalytic reaction but asparagine is used to stabilise the structure.

Other use of β-glucosidases
β-glucosidase is now used for the synthesis of biofuel. Wood is an abundant and renewable energy which can be changed into bioethanol thanks to enzymatic hydrolysis. This synthesis needs five steps. First it is pre-hydrolysis. The structure is divided into lignin and (hemi)cellulose. The cellulase can better access the structure to act on it. The second step: hydrolysis is the most important. A cellulase is a complex of 3 enzymes which act together to hydrolyse cellulose: Endoglucanase breaks the chain in the middle of the molecular structure of cellulose. Exoglucanase binds an available end of the chain and isolates it. Then units of cellobiose are cut (two units of glucose which are together). To finish, β-glucosidase divides cellobiose into two glucoses. When they ferment, they become ethanol. The final product is obtained thanks to fermentation, distillation and deshydratation.

Additional Resources
For additional information, see: Carbohydrate Metabolism

3D structures of Beta-glucosidase
Update June 2011

3ahx – GBA – Clostridium cellulovorans

3ahy – GB – Trichoderma reesei

3abz – KmGB – Kluyveromyces marxianus

2x40 – TnGB3B – Thermotoga neapolitana

3gno - JrGB residues 38-521 – Japanese rice

2rgl, 2rgm - JrGB residues 29-504

3f4v, 3f5j, 3f5k, 3f5l - JrGB7 (mutant)

3ahz – tGB – termite

3fiy, 3cmj - UbGB catalytic domain (mutant) – Uncultured bacteria

2o9p, 1bga – PpGBB – Paenibacillus polymyxa

2jfe - hGB cystolic – human

2e3z – PcGB – Phanerochaete crysosporium

2dga - wGB residues 1-520 – wheat

2vff, 1vff – GB – Pyrococcus horikoshii

1oif, 1od0 – TmGB catalytic domain - Thermotoga maritima

1ug6 – GB - Thermus thermophilus

1hxj, 1e1e – ZmGB – Zea mays

1e4l - ZmGB (mutant)

1gon – SsGB – Streptomyces sp.

1qox – GB - Bacillus circulans

1tr1 - BpGBB (mutant) - Bacillus polymyxa

1cbg – GB cyanogenic – White clover

3aiu - rGB residues 50-568 – rye

3f93, 3f94 – PsGB residues 28-840 – Pseudoalteromonas

3f95 – PsGB residues 657-840

3ptk – rGB - rice

Beta-glucosidase complex with sugar
3ptm, 3ptq – rGB + glucoside

3ai0 – tGB + glucoside

3ac0 - KmGB + glucoside

2x41, 2x42 - TnGB + glucoside

3air – wGB residues 50-569 + glucoside + dinitrophenol

3ais - wGB residues 50-569 (mutant) + glucoside + aglycone

3aiq - wGB residues 50-569 + aglycone

3aiv - rGB residues 50-568 + aglycone

3aiw - rGB residues 50-568 + glucoside + dinitrophenol

3gnp, 3gnr - JrGB residues 38-521 + glucoside

3aht, 3ahv – JrGB7 (mutant) + saccharide

1oin - TmGBA + glucoside

3fiz, 3fj0 - UbGB residues 18-482 (mutant) + glucoside

2zox, 2e9l, 2e9m - hGB cystolic + glucoside

2o9s, 2o9r, 2z1s – PpGBB + saccharide

2o9t - PpGBB + glucoside

1uyq - PpGBB (mutant) + glucoside

1bgg - PpGBB + gluconate

2jie - BpGBB + glucoside

1e4i - BpGBB (mutant) + glucoside

2e40 – PcGB + gluconolactone

1v08 – ZmGB + gluco-tetrazole

1e1f - ZmGB + glucoside

1h49, 1e4n, 1e56 - ZmGB (mutant) + aglycone

1gnx - SsGB + saccharide - Sulfolobus solfataricus

Beta-glucosidase complex with inhibitor
2wbg, 2wc3, 2wc4, 2vrj, 2jal, 2j75, 2j77, 2j78, 2j79, 2j7b, 2j7d, 2j7e, 2j7f, 2j7g, 2j7h, 2j7c, 2ces, 2cet, 2cbu, 2cbv, 1uz1, 1wj3, 1oim – TmGBA + inhibitor

2cer – SsGB + inhibitor

1e55 - ZmGB (mutant) + inhibitor

6-phospho-β-glucosidase
1s6y – PGB – Geobacillus stearothermophilus

1up4 – TmPGB

1up6, 1up7 – TmPGB + NAD + G6P

3qom – PGB – Lactobacillus plantarum

1h4p – PGB I/II – yeast

3eqn – WfPGB – White-rot fungus

3eqo – WfPGB + glucolactone

Glucan 1,3-β-glucosidase
3n9k – CaGGB + glucoside – Candida albicans

3o6a – CaGGB (mutant)